Lymphocyte population in blood is defined by a number of subclasses which play distinct roles in the immune response. For example, the relative number of lymphocytes in various subclasses is likely to change in disease states. Hence, enumeration and identification of cells of the various subclasses yields an indication not only of the constituency of the blood in particular, but generally with respect to the relative well being of the organism.
It is known that at least several particular subclasses of functionally distinct lymphocytes can be identified on the basis of antigenic determinants on the cell surface. In particular, considerable academic and clinical interest has occurred for T-lymphocytes. Among others, T-lymphocytes are characterized by particular, identifiable antigenic determinants on their surface, which distinguish the cells of that subclass from other blood cells and from the cells of other lymphocyte subclasses. Hence, interest is high in identifying and producing reagents which include antibodies which are selectively reactive with the lymphocyte subclasses of interest.
It should be understood that there are two principal classes of lymphocytes involved in the immune system of humans and animals. The first of these (the thymusderived cell or T cell) is differentiated in the thymus from haemopoietic stem cells. While within the thymus, the differentiating cells are termed "thymocytes." The mature T cells emerge from the thymus and circulate between the tissues, lymphatics, and the bloodstream. These T cells form a large proportion of the pool of recirculating small lymphocytes. They have immunological specificity and are directly involved in cell-mediated immune responses (such as graft rejection) as effector cells. Although T cells do not secrete humoral antibodies, they are sometimes required for the secretion of these antibodies by the second class of lymphocytes discussed below. Some types of T cells play a regulating function in other aspects of the immune system. The mechanism of this process of cell cooperation is not yet completely understood.
The second class of lymphocytes (the bone marrow-derived cells or B cells) are those which secrete antibody. They also develop from haemopoietic stem cells, but their differentiation is not determined by the thymus. In birds, they are differentiated in an organ analogous to the thymus, called the Bursa of Fabricius. In mammals, however, no equivalent organ has been discovered, and it is thought that these B cells differentiate within the bone marrow.
It is now recognized that T cells are divided into at least several subtypes, termed "helper", "suppressor", and "killer" T cells, which have the function of (respectively) promoting a reaction, suppressing a reaction, or killing (lysing) foreign cells. These subclasses are well understood for murine systems, but they have only recently been described for human systems. See, for example, R. L. Evans, et al., Journal of Experimental Medicine, Volume 145, 221-232, 1977; and L. Chess and S. F. Schlossman--"Functional Analysis of Distinct Human T Cell Subsets Bearing Unique Differentiation Antigens", in "Contemporary Topics in Immunobiology", O. Stutman, Editor, Plenum Press, 1977, Volume 7, 363-379.
The ability to identify or suppress classes or subclasses of T cells is important for diagnosis or treatment of various immunoregulatory disorders or conditions.
For example, certain leukemias and lymphomas have differing prognosis depending on whether they are of B cell or T cell origin. Thus, evaluation of the disease prognosis depends upon distinguishing between these two classes of lymphocytes. See, for example, A. C. Aisenberg and J. C. Long, The American Journal of Medicine, 58:300 (March, 1975); D. Belpomme, et al., in "Immunological Diagnosis of Leukemias and Lymphomas", S. Thierfelder, et al., eds, Springer, Heidelberg, 1977, 33-45; and D. Belpomme, et al., British Journal of Haematology, 1978, 38, 85. Certain disease states (e.g. juvenile rheumatoid arthritis and certain leukemias) are associated with an imbalance of T cell subclasses. It has been suggested that autoimmune diseases generally are associated with an excess of "helper" T cells or a deficiency of certain "suppressor" T cells, while malignancies generally are associated with an excess of "suppressor" T cells. In certain leukemias, excess T cells are produced in an arrested stage of development. Diagnosis may thus depend on the ability to detect this imbalance or excess. See, for example, J. Kersey, et al., "Surface Markers Define Human Lymphoid Malignancies with Differing Prognoses" in Haematology and Blood Transfusion, Volume 20, Springer-Verlag, 1977, 17-24, and references contained therein. Recently, monoclonal antibody techniques have been utilized to produce large quantities of highly purified antibody to various lymphocyte subclasses. Utilizing such antibodies, it has proven feasible to assay an individual's lymphocytes to determine the relative numbers of cells in various subclasses. Further, utilizing direct or indirect techniques, the antibodies may be fluorescently tagged, thereby rendering the samples under consideration amenable to flow cytometric analysis.
Conventional immunofluorescence techniques presently include the physical isolation of the lymphocytes from other leukocytes as a preliminary step. This step eliminates the possibility that non-specifically stained monocytes or granulocytes might be counted as specifically stained lymphocytes. This initial lymphocyte isolation step is long and arduous, however, in fact much longer than the relatively simple steps of staining or analyzing the lymphocytes. Clearly, in the clinical arena, wherein there exists a considerable premium on rapid, repetitive analyses, the necessity of isolating the lymphocytes from other leukocytes is a virtually insurmountable impediment. Furthermore, even for those research applications wherein time is at less of a premium, the initial lymphocyte isolation step involves the risk of loss of some lymphocytes, during removal of monocytes and granulocytes, which introduces uncertainty and inaccuracy to the subsequent analysis. Correspondingly, failure completely to eliminate other blood cells from the lymphocytes to be isolated introduces considerable risk of error, and the presence of such other cells may well cause errors in the subsequent analysis.
It is, accordingly, a primary object of the present invention to provide a method and apparatus for identifying and enumerating specific subclasses of lymphocytes, while avoiding the necessity for prior separation of lymphocytes from other blood cells. It is an associated object to provide such methods and apparatus for other types of blood cells which involve similar antigenic characteristics.
Correspondingly, it is an object to provide methods and apparatus which are considerably faster than present techniques, and which substantially obviate faulty analysis attendant to artifact data from other cells, or loss of data through loss of lymphocytes from the sample. Further, it is an object to provide such methods and apparatus wherein the speed and relative simplicity involved renders lymphocyte subclass identification and enumeration a viable clinical tool.